Innate immunity is the first line of defense against microbes and it is indispensable in preventing infections as well as in the development and regulation of the adaptive immune system. Innate immunity is based on the ability of genome-encoded proteins to recognize and bind microbial surface structures, which is followed by the activation of the innate immune response via various cell signaling pathways.

Tumor necrosis factor receptor (TNFR) signaling and cytokine release are strictly regulated and essential for a normal immune response. However, in certain diseases, like infections and autoimmune diseases, cytokines are produced in excess, which prolongs the inflammation and causes tissue damage. Clinical medicine is trying to prevent cytokine overproduction by suppressing TNFR signaling. However, this is challenging, since despite the extensive research carried out in this field in recent years, the mechanisms and regulation of TNFR signaling are not thoroughly understood.

The pathways of innate immunity signaling are evolutionarily conserved from insects to humans. Unlike mammals, insects have no adaptive immunity, and hence they are completely dependent on their innate immune response. For these reasons the fruit fly (Drosophila melanogaster) is a suitable model organism for studying innate immunity. In Drosophila, the immune response to Gram-negative bacteria is mediated mainly via the Imd (immune deficiency) signaling pathway, whose intracellular parts resemble the mammalian TNFR signaling pathway. The aim of this research project was to identify and molecularly characterize the components of the Imd pathway and regulatory proteins using a large-scale RNA interference (RNAi) screen. The function and importance of three of the identified regulators, namely Tak1-associated binding protein 2 (Tab2), Inhibitor of apoptosis 2 (Iap2), and Poor Imd response upon knock-in (Pirk), were then further assessed. Using Drosophila S2 cells we showed that Tab2 is essential for both the early and sustained immune responses, while Iap2 mainly regulates the sustained immune response. In addition, we discovered that when Iap2 was removed from fruit flies by in vivo RNAi the flies became susceptible to Gram-negative bacterial infections.

Pirk was a previously unknown protein that we demonstrated could suppress the Imd pathway activity both in S2 cells and in flies. The inhibitory action of Pirk was shown to be efficient enough to sensitize the flies to Gram-negative bacterial infections. We found that Pirk interacts with the receptor of the Imd pathway, PGRP-LC (Peptidoglycan recognition protein LC), and the downstream component IMD. However, the elucidation of the exact mechanism of Pirk action requires further studies.

The present study demonstrates that Imd signaling is strictly regulated and more complex than was previously thought. Drosophila as a model organism provides tools to efficiently study innate immunity. In addition, the results gained from this research in flies can provide new perspectives and may help understand also the mechanisms of signaling in the mammalian innate immune system.